TempO-Seq Gene Expression Profiling of Intracellular Stained FACS Sorted Cells

DESCRIPTION provided by applicant We propose to develop an intracellular stained Fluorescence Activated Cell Sorter Templated Oligonucleotide Sequencing whole transcriptome gene profiling assay icsFACS TempO Seq addressing an unmet need by enabling quantitative gene expression analysis of fixed permeabilized and intracellular stained and sorted cells a method used to purify many different functionally important and rare subsets of cells The assay will have the sensitivity to measure gene expression from small sets of cells and from single fixed and sorted cells whose function fate or lineage are identified by cytosolic or nuclea biomarkers including immune cells and circulating or tissue dissociated stem and cancer cells Having shown that TempO Seq measurement is unimpaired by fixation that antibody staining can be integrated into the TempO Seq protocol and that high quality data is obtained at the single cell level using RNA titrations we propose to leverage a separately funded effort to implement a whole transcriptome TempO Seq assay to develop and prove the feasibility of a whole transcriptome icsFACS TempO Seq assay TempO Seq effectively reduces the sequencing cost sample to andlt $ transcriptome at third party vendor rates compared to RNA Seq which is typically several hundred dollars up to $ HiSeq per transcriptome depending on the depth of sequencing Investigators can measure a focused set of genes of fewer than genes up to the whole transcriptome plus measure specific mutations or gene fusions with icsFACS TempO Seq We will implement the assay determine its performance sensitivity reproducibility dynamic range and then perform gene expression profiling of human T cell subsets using anti CD activated human peripheral blood lymphocytes which are stained for a combination of surface CD and intracellular TBX eomesodermin Foxp antigens and then FACS sorted Analysis of single cell preps and small homogeneous populations will generate important novel data with single cell gene expression being used to define additional molecular phenotypes reveal molecular pathways and provide insight in the overall regulation of single cell transcription Based on this feasibility the commercial development validation and launch of icsFACS TempO Seq will be carried out in Phase II Flow cytometry and FACS sorting remains a robust and growing market into which BioSpyder will sell icsFACS TempO Seq kits No specialized hardware is required to perform the assay nor is extensive bioinformatics required for data analysis making this assay easy to adopt PUBLIC HEALTH RELEVANCE We propose to develop and demonstrate the feasibility and utility of an intracellular stained Fluorescence Activated Cell Sorter icsFACS Templated Oligonucleotide Sequencing icsFACS TempO Seq whole transcriptome gene expression profiling assay to monitor gene expression from fixed permeabilized and intracellularly stained and sorted cells icsFACS TempO Seq will address an unmet need within the large and growing flow cytometry market to profile expression of focused sets of genes up to the whole transcriptome in a cost effective and easy to adopt manner from subpopulations and single cells that can only be identified using intracellular staining of biomarkers such as cells critical in te immune response pathway cancer and other diseases We will demonstrate the feasibility of profiling gene expression from single cells and small populations to define molecular phenotypes and pathways of activation and differentiation using anti CD CD activated human T cell subsets defined using intracellular antibody staining of the transcription factors TBX eomesodermin and Foxp respectively and surface staining for a combination of markers CD generating novel data regarding the molecular phenotypes and mechanisms of T cell subsets that can only be identified using intracellular staining and of individual cells within those subsets